Extraction of the mother and recombinant plasmids:

The plasmid extraction was carried out with DNA-spin™ Plasmid DNA Purification Kit, iNtRON Biotechnology Inc., South Korea according to manufacturer ̓s instructions.

DNA purification of phbB and phbC genes after electrophoresis:

The obtained specific bands after PCR amplification responsible for phbB and phbC genes were purified by MEGAquick-spin™ Plus Total Fragment DNA Purification Kit, iNtRON Biotechnology Inc., South Korea

Restriction digestion of phbB and phbC genes fragments and plasmid pET-29a(+):

Purified phbB and phbC genes and isolated pET-29a(+) plasmid were subjected to restriction digestion reaction with BamHI and XhoI restriction enzymes (New England BioLab, USA). According to the manufacturer's instructions, 50 U of each NEB enzyme, 1x of NE Buffer 4 (recommended buffer for double digestion of BamHI and XhoI), 10µg DNA (fragments and plasmid) were used in a final volume of 100µL, and incubated at 37°C for 2 h. After incubation, the digested fragments were purified using PCR cleanup protocol (Gel extraction protocol without step of agarose gel running), and the digested plasmid was electrophoresed on 1.5% agarose gel and cut under long wave UV light, and gel slice was subjected to gel purification.

Ligation of phbB and phbC fragments with plasmid pET-29a(+):

Recovered phbB and phbC genes were ligated with the linearized vector pET-29a(+) at BamHI and XhoI recognition sites, using T4 DNA ligase (New England BioLab, USA). Ligation reaction was carried out in 20 µL volume with the vector insert ratio (1:1 , 1:3 and 1:5) as follow: 500 U of T4 ligase, 1X ligation buffer with ATP (50mM Tris-HCl, 10mM MgCl₂, 10mM DTT, 1 mM ATP, 25µg/mL BSA), ≈10ng of the DNA (plasmid and insert), and incubated overnight at 16°C.

Competent cell (E. coli DH5α) preparation:

Competent cells were prepared using Calcium-Chloride according to the method described by Sambrook and Russell²⁸.

Bacterial transformation:

The introduction of ligation mixture of pET-29a(+) with phbB and phbC genes to E. coli DH5α was done by heat shock. Ten microliters of ligation mixture was added to 100µL of CaCl₂-treated competent cells, mixed by tapping and stored on ice for 20 min. Then, the mixture was subjected to 42°C for 90 sec and immediately transferred to ice for 2-5 min followed by addition of 800 µL of pre-warmed LB medium and incubated at 37°C with slow shaking. Different aliquots of these transformed competent cells were spread on LB/kanamycin plates. When plates were dried, inverted and incubated overnight at 37°C (All steps were done under aseptic conditions).

Screening for the positive colonies after transformation:

Colony-PCR technique was used for screening the transformed positive colony that has the phbB and phbC genes. PCR amplification was carried out using pET- phbB and phbC specific primers. Colonies that at least 1 mm in diameter was picked for screening into 25 µL of PCR reaction mixture, which prepared as follows: 0.25 µL of Taq polymerase (5U/µL), 2.5µL of 10x Taq buffer, 0.25µL of dNTPs (50mM for each), 0.5µL (10 pmol/µL) of each primers; using the following parameters: 94°C pre-denaturation 5 min, followed 35 cycles of 40 sec denaturation 94°C, 40 sec annealing 57°C, 1.5 min extension 72°C, and 10 min final extension 72°C. Then, an aliquot of this amplification was visualized on 1% agarose gel. Sequences of pET specific primers and resulted PCR products, the first pair was forward primer (pET-F) 5'-CGTCCGGCGTAGAGGATC-3' and the reverse primer (pET-R) 5'-ATCCGGATATAGTTCCTCCTTTC-3'. The resulting PCR products included the insert gene plus 290bp from the mother pET-29a(+) plasmid.

Transformation of E. coli BL21 (DE3) pLysS and DNA sequencing:

The positive transformed colony of E. coli DH5α was subjected to plasmid isolation using with DNA-spin™ Plasmid DNA Purification Kit, (iNtRON Biotechnology Inc., South Korea) and the recombinant pET-29a(+) plasmids were transformed into expression host E. coli BL21 (DE3) pLysS (Novagen, Germany) using calcium chloride protocol that described previously with DH5α transformation. In the same manner, the positive transformed colony was screened by colony PCR technique as above. The sequencing was done on both the DNA strands by universal pET-29a(+) forward and Reverse primers using ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Germany).

Expression mechanism of phbB and phbC genes in BL21 under the T7 promoter:

A single colony of BL21 (DE3) harboring the recombinant plasmid was inoculated in 5mL of LB/kanamycin and incubated at 37°C with 250 rpm shaking overnight. The fresh overnight cultures were inoculated in LB/kanamycin at a dilution of 1:100 and grown at 25°C with 250 rpm shaking until the absorbance at 600 nm (OD600) reached 0.3. After that, the cultures were induced with isopropyl-b-D-thiogalactopyranoside (IPTG, Merck) at a final concentration of 0.5mM and were further incubated at 25°C with 200rpm shaking for 4 h before being harvested by centrifugation at 8000rpm for 10 min.

Tricine method for recombinant protein electrophoresis:

Total protein preparation:

Total protein by tricine electrophoresis were set up by centrifugation of 1mL of each culture (IPTG-induced and un-induced) and cell pellets were re-suspended in 20 µL of refined water and 20µL of sample buffer (100 mM Tris-HCl pH 6.8, 4% w/v SDS, 20% v/v glycerol, 0.02% w/v bromophenol blue, 10% v/v β-mercaptoethanol) was added and boiled for 3 min.

Gels preparation:

Solutions for making gels were prepared according to Schägger and Von Jagow²⁹. The separating gel was 12% T, 3% C and the stacking gel was 4% T, 3% C. All solutions were stored at 4 °C.

Run conditions:

Electrophoresis was performed at room temperature using a voltage stepped procedure: voltage was kept constant (30 V) until the samples completely left the stacking gel and then the voltage were increased 15 V per min for 4 times. Voltage was maintained constant (90-100 V) until the tracking dye reached the bottom of the gel.

Fixing, staining and de-staining:

Immediately after ending electrophoresis, gels were removed from the plates and placed in a fixation solution. After 30 min, the fixation solution was replaced by a staining solution, where gels were left for 30 min at 50°C³⁰. De-staining of gels was accomplished by heating in a microwave oven during short periods and making several changes of distilled water during the de-staining procedure.

RESULTS:

Amplification and molecular detection of phbB and phbC genes after genomic DNA extraction:

The bacterial genomic DNA from Bacillus aryabhattai 6N-NRC strain was extracted and separated by agarose gel electrophoresis as shown in Fig. 1a. The obtained results indicated that, two genomic DNA samples of Bacillus aryabhattai 6N-NRC strain lanes (1 and 2) gave one band upper 10 kb without any smear DNA. So, the above result exhibited that the genomic DNA was highly purified and un-fragmented. The phbB and phbC genes were detected and amplified by direct PCR. Fig.1 (b, c) showed that the obtained PCR products representing the phbB and phbC genes were approximately 744 bp and 1089bp, respectively.

a) b) c)

Fig. 1(a-c): (a) Extraction and detection of the complete bacterial genomic DNA of Bacillus aryabhattai 6N-NRC strain (lanes 1 and 2). Lane: M, DNA size marker (iNtRON Biotechnology Inc., South Korea). (b) Agarose gel (1%, w/v) showing PCR amplified profile of Bacillus aryabhattai 6N-NRC phbB gene (lane 1). Lane: M, DNA size marker (iNtRON Biotechnology Inc., South Korea). (c) Agarose gel (1%, w/v) showing PCR amplified profile of Bacillus aryabhattai 6N-NRC phbC gene (lane 1). Lane: M, DNA size marker (iNtRON Biotechnology Inc., South Korea).

Cloning of purified pET-29a(+) vector and phbB and phbC genes:

Plasmid vector was extracted and purified from the E.coli containing pET-29a(+) by DNA-spin™ Plasmid DNA Purification Kit, iNtRON Biotechnology Inc., South Korea. The purified pET-29a(+) plasmid vector was used for cloning of phbB and phbC genes obtained from Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1), the recombinant vector was applied for studying the genes responsible for the production of polyhydroxybutyrate (PHB). The double restriction digestion reaction of pET-29a(+) plasmid vector using restriction enzymes BamHI and XhoI was carried out to prepare the linear vector then the linear vector was purified by PCR cleanup kit. The purified phbB and phbC genes were also subjected to double restriction digestion reaction with BamHI and XhoI restriction enzymes (New England BioLab, USA).

After restriction digestion and purification by PCR cleanup kit of phbB and phbC genes, the ligation of phbB and phbC genes with plasmid pET-29a(+) was done using T4 DNA ligase (New England BioLab, USA). Then the ligated recombinant vectors were transformed into E. coli DH5ɑ competent cells and the E. coli DH5ɑ carrying the recombinant vectors with phbB and phbC inserts was resistant to 50 µg/ml of kanamycin. The recombinant vectors containing phbB and phbC genes and the purified linear vector after extraction from E. coli DH5ɑ competent cells were loaded to agarose gel to examine their molecular weights.

The obtained results were illustrated in Fig. 2a indicated that, the mother pET-29a(+) plasmid lane (1) gave one band upper 10 kb but the recombinant vectors containing phbB and phbC genes gave two bands. When comparing with the band of plasmid pET-29a(+) lane (1), it was found that the level of the bands of the recombinant vectors containing phbB and phbC genes (lanes 2 and 3) were higher than the mother plasmid (lane 1), and also the bands were found in recombinant vector containing phbC gene (lane 3) was higher than recombinant vector containing phbB gene (lane 2), due to the large phbC gene size in comparison with phbB gene.

Confirmation of the successful cloning in E. coli DH5α:

The DNA was extracted from E. coli DH5α colonies containing recombinant pET29a-phbB and pET29a-phbC plasmids. The purified construct was tested by carrying out PCR reaction to amplify the fragment comprising the cloning site of phbB and phbC genes against DNA ladder. The PCR analyses of two recombinant E. coli DH5α strains containing pET29a-phbB and two recombinant E. coli DH5α strains containing pET29a-phbC were shown in Fig. 2(b, c). After gel was examined using UV trans-illuminator it showed that the phbB gene amplified by PCR showed strong band on 744 bp as shown in Fig. 2b, while that phbC demonstrated positive amplification at molecular size 1089 bp as shown in Fig. 2c. The obtained results suggested that the desired genes are presented in genetically engineered strains of E. coli DH5α. Also, the genes were of similar molecular weight, as previous studies reported.

(a) (b) (c)

Fig. 2(a, b, c): (a) Agarose gel electrophoresis for of pET-29a(+) plasmid (lane 1), pET29a-phbB (lane 2) and pET29a-phbC (lane 3) constructs obtained after transformation of E.coli DH5α strain against DNA size marker (Lane: M) (iNtRON Biotechnology Inc., South Korea). Colony-PCR for (b) pET29a-phbB, (c) pET29a-phbC constructs in DH5α strain (lanes 1 and 2). Lane M, DNA size marker (iNtRON Biotechnology Inc., South Korea).

Propagation of pET29a-phbB and pET29a-phbC in E. coli BL21:

Recombinant pET-29a(+) plasmid isolated from E. coli DH5α and harboring phbB and phbC genes were transformed into E. coli BL21 competent cells against a negative control, then spread over LB/kanamycin media plate. The obtained results after the plates were incubated overnight exhibited that no colony in LB/kanamycin media plate spread with the E. coli BL21 competent cells, because the E. coli BL21competent cells did not harbor pET-29a(+) plasmid carrying the kanamycin resistant gene. While, the E. coli BL21 competent cells transformed with pET29a-phbB and pET29a-phbC gave many colonies in LB medium containing 50 µg/ mL of kanamycin. So, the E. coli BL21 competent cells transformed with pET29a-phbB and pET29a-phbC acquired kanamycin resistance.

Confirmation of successful cloning in E. coli BL21:

DNA was extracted from the E. coli BL21 colonies containing recombinant pET29a-phbB and pET29a-phbC plasmids by Colony-PCR technique. The purified construct was tested by carrying out PCR reaction to amplify the fragment comprising the cloning site of phbB and phbC genes against DNA ladder. The PCR analyses of the two recombinant E. coli BL21 strains containing pET29a-phbB and the three recombinant E. coli BL21 strains containing pET29a-phbC were shown in Fig. 3(a, b). After gel was examined using UV- transilluminator, it showed strong band with molecular size 744 bp for phbB gene and another band with molecular size 1089 bp for phbC gene. The obtained results suggested that the desired genes are presented in genetically engineered strains of E. coli BL21. Also, the genes were of the same molecular weights as reported by the previous studies.

(a) (b)

Fig. 3(a, b): Colony-PCR for (a) pET29a-phbB, (b) pET29a-phbC constructs in E. coli BL21 strain (lanes 1 and 2). Lane M, DNA size marker (iNtRON Biotechnology Inc., South Korea).

Amplification the phbB and phbC genes using specific primers of pET-29a(+) plasmid:

Colony-PCR was performed for bacterial colonies grown on antibiotic (kanamycin) containing medium and the plasmid specific primers to detect the right position of the genes on the plasmid. The colony of pET29a-phbB showed positive amplification at 1034bp with plasmid specific primers likewise, colony of pET29a-phbC demonstrated positive amplification at molecular size 1379bp with the plasmid specific primers (Fig. 4a, b). This means that the genes were at the optimal size plus 290 bases of the mother plasmid vector as previous studies decided. These obtained fragments can be used to perform DNA sequencing analysis so that the sequence of the complete gene can be obtained without any deficiency.

(a) (b)

Fig. 4(a, b): PCR products for (a) pET29a-phbB, (b) pET29a-phbC constructs in E. coli BL21 strain with plasmid specific primers (lane 1). Lane M, DNA size marker (iNtRON Biotechnology Inc., South Korea.

DNA sequencing of phbB and phbC genes using specific primers of pET29a(+) plasmid:

The recombinant plasmids (pET29a-phbB and pET29a-phbC) were extracted and purified by using DNA-spin™ Plasmid DNA Purification Kit, iNtRON Biotechnology Inc., South Korea. The sequencing was done on both the DNA strands by universal pET-29a(+) forward and reverse primers using ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems). The phbB sequence is shown in Fig. 5a and the open reading frame consisted of 744 bp. Sequences obtained were analyzed for variability or homogeneity through NCBI Blast and the phylogenetic tree was drawn as shown in Fig. 5b. The DNA sequence of phbB was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1) and B. megaterium (GenBank accession no. CP026736.1). On the other hand, The phbC sequence is shown in Fig. 5c and the open reading frame consisted of 1089 bp. Sequences obtained were analyzed for variability or homogeneity through NCBI Blast and the phylogenetic tree was drown as shown in Fig. 5d. The DNA sequence of phbC was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (GenBank accession no. CP024035.1) and B. megaterium (GenBank accession no. CP026736.1).

(a)

(b)

Fig. 5(a, b): (a) Phylogenetic tree of phbB in comparison with other sequences (NCBI Neighbor Joining), (b) Phylogenetic tree of phbC in comparison with other sequences (NCBI Neighbor Joining).

Analysis of the recombinant proteins by Tricine- Polyacrylamide gel electrophoresis:

Colonies of pET29a-phbB and pET29a-phbC were sampled just before induction with isopropyl-β-D-thiogalactopyranoside (IPTG) for 8 hours against negative controls of E. coli BL21(DE3strains) containing the mother pET-29a plasmid. After induction, the samples were loaded to tricine SDS-PAGE gel. Tricine SDS-PAGE of the expressed phbB gene in E. coli BL21 strain showed distinct band of intensity at 26.3 KD as shown in Fig. 6a. The obtained results suggested that the desired protein band is presented in genetically engineered strains of E. coli BL21. Also, the protein band was of the same molecular weight as previous studies had reported.

Furthermore, tricine SDS-PAGE was applied on the pET29a-phbC colonies against negative controls of E. coli BL21(DE3strains) containing the mother pET-29a plasmid Fig. 6b, the obtained results showed that the negative controls have no distinct band at 37.5 KD position while, the expressed phbC gene in E. coli BL21 strain exhibited distinct band of intensity at 37.5 KD. These results confirmed that the desired protein band is presented in genetically engineered strains of E. coli BL21 and the protein band was of the ideal molecular weight.

(a) (b)

Fig. 6(a, b): Tricine- Polyacrylamide gel electrophoresis for testing the expression of (a) phbB gene, (b) phbC gene in E. coli BL21. Lane: M, protein marker (Chromatein Prestained Protein Ladder, Vivantis, PR0602); C, non-induced BL21strain whish containing the mother pET-29a plasmid as a control sample; NB1, un-induced pET29a- phbB in BL21; NB2, induced pET29a- phbB in BL21.

DISCUSSION:

Many researchers are concerned with genetic engineering in different fields^31-35. Polyhydroxyalkanoate (PHA) biosynthesis genes have been cloned and expressed in recombinant E. coli as an ideal strategy for cost effective biopolymer synthesis^36,4. This study was conducted to characterize some responsible genes for PHB production in Bacillus aryabhattai 6N-NRC strain (GenBank accession no. MH997667.1). Initially, the genomic DNA was isolated from the superior Bacillus aryabhattai 6N-NRC strain. It was established that the isolated DNA amount was sufficient and pure to carry out subsequent studies according the agarose gel electrophoresis and spectrophotometer experiments on the isolated DNA samples. Furthermore, the phbB and phbC genes were detected and amplified by direct PCR. The PCR products representing both phbB and phbC genes were with molecular weights 744 bp and 1089 bp, respectively. These results were compatible with many previous studies by Li et al.³⁷ and Rehman³⁸ where they confirmed the same molecular weights of the phbB and phbC genes. On the other hand, Galehdari et al.³⁹ detected different molecular weight of the phbC gene (1704 bp) when separately amplified the phbC gene from Azotobacter genome.

The important stage in this work was to load the phbB and phbC genes into the pET-29a(+) plasmid vector and then make a genetic transformation of Escherichia coli and determine the occurrence of the genetic transformation with the recombinant vector. The genetic transformation process was carried out in two phases. First the genetic transformation of E. coli DH5α was carried out for the purpose of multiplication of the plasmid then the genetic transformation of E. coli BL21 was carried out with the aim of genetic expression of the phbB and phbC genes under study. After the genetic transformation experiments of E. coli DH5α and E. coli BL21, it was confirmed that the pET-29a(+) carrying the phbB and phbC genes were presented, detected by colony PCR technique and the transformed E. coli DH5α and E. coli BL21 were positive to grow on LB/kanamycin media. These results confirmed the success of genetic transformation and transfer of genes under study and are also consistent with many previous studies, such as Li et al.⁴⁰ and Wu et al.²¹.

Moreover, the amplification the phbB and phbC genes using specific primers of pET-29a(+) plasmid showed that the colonies of pET29a-phbB found positive amplification at 1034bp with plasmid specific primers and colonies recorded in plate pET29a-phaC demonstrated positive amplification at molecular size 1379bp with the plasmid specific primers. This means that the genes were at the optimal size plus 290 bases of the mother plasmid vector as previous studies decided and the obtained fragments can be used to perform DNA sequencing analysis to sequence the complete genes under study without any deficiency. Furthermore, the sequencing was done on both the DNA strands by universal pET-29a(+) forward and reverse primers using ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems, Germany). The open reading frame of phbB sequence consisted of 744 bp and found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of Bacillus aryabhattai (GenBank accession no. CP024035.1) and Bacillus megaterium (GenBank accession no. CP026736.1),while the open reading frame of phbC sequence consisted of 1089 bp and found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of Bacillus aryabhattai (GenBank accession no. CP024035.1) and Bacillus megaterium (GenBank accession no. CP026736.1). These results are consistent with the previous studies^{11,41,42,43,44}.

Finally, the analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine- polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands at intensity of 26.3 KD and 37.5 KD respectively. The obtained results suggested that the desired protein bands are presented in genetically engineered strains of E. coli BL21. Also, the protein bands were of the same molecular weight, as previous studies had decided according to Tanaka et al.⁴⁵.On the other hand, Shi et al.⁴⁶ found differences in molecular weights (27KD and 64 KD) of the phbB and phbC bands, respectively.

CONCLUSION:

This investigation has revealed that the PCR techniques are effective molecular methods for amplification of phbB and phbC genes which are responsible for PHB production and these targeted genes were isolated and purified. The PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a(+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. Recombinant strains have been successfully constructed to help in subsequent studies resulting in the increased production of PHB biopolymer.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Eerkes-Medrano D, Thompson RC, Aldridge DC. Microplastics in freshwater systems: a review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. Water Research. 2015; 75: 63-82.‏

2. Jain R, Tiwari A. Biosynthesis of planet friendly bioplastics using renewable carbon source. Journal of Environmental Health Science and Engineering. 2015; 13(1): 1-5.‏

3. Lusher AL, Burke A, O’Connor I, Officer R. Microplastic pollution in the Northeast Atlantic Ocean: validated and opportunistic sampling. Marine Pollution Bulletin. 2014; 88(1-2): 325-333.‏

4. Pillai AB, Kumar AJ, Kumarapillai H. Enhanced production of poly (3-hydroxybutyrate) in recombinant Escherichia coli and EDTA–microwave-assisted cell lysis for polymer recovery. AMB Express. 2018; 8(1): 142.‏

5. Hang X, Zhang G, Wang G, Zhao X, Chen GQ. PCR cloning of polyhydroxyalkanoate biosynthesis genes from Burkholderia caryophylli and their functional expression in recombinant Escherichia coli. FEMS Microbiology Letters. 2002; 210(1): 49-54.‏

6. Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). Journal of Biological Chemistry. 1989; 264(26): 15298-15303.‏

7. Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase. Journal of Biological Chemistry. 1989; 264(26): 15293-15297.‏

8. Steinbüchel A, Schlegel HG. Physiology and molecular genetics of poly (β‐hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus. Molecular Microbiology. 1991; 5(3): 535-542.‏

9. Rehm BH. Polyester synthases: natural catalysts for plastics. Biochemical Journal. 2003; 376(1): 15-33.‏

10. Schubert P, Steinbüchel A, Schlegel HG. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. Journal of Bacteriology. 1988; 170(12): 5837-5847.‏

11. Slater SC, Voige WH, Dennis DE. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. Journal of Bacteriology. 1988; 170(10): 4431-4436.‏

12. Liebergesell M, Steinbüchel A. Cloning and nucleotide sequences of genes relevant for biosynthesis of poly (3‐hydroxybutyric acid) in Chromatium vinosum strain D. European Journal of Biochemistry. 1992; 209(1): 135-150.

13. ‏Schembri MA, Bayly RC, Davies JK. Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. FEMS Microbiology Letters. 1994; 118(1-2): 145-152.‏

14. Liebergesell M, Steinbüchel A. Cloning and molecular analysis of the poly (3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea. Applied Microbiology and Biotechnology. 1993; 38(4): 493-501.‏

15. Hustede E, Steinbüchel A. Characterization of the polyhydroxyalkanoate synthase gene locus of Rhodobacter sphaeroides. Biotechnology Letters. 1993; 15(7): 709-714.‏

16. Valentin HE, Steinbüchel A. Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene. Applied Microbiology and Biotechnology. 1993; 39(3): 309-317.‏

17. Pieper U, Steinbüchel A. Identification, cloning and sequence analysis of the poly (3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber. FEMS Microbiology Letters. 1992; 96(1): 73-79.‏

18. El Rabey HA, Albureikan MO, Aly MM, Kabli SA, Schneider K, Nolke G. Isolation, cloning and sequencing of poly 3-hydroxybutyrate synthesis genes from local strain of Bacillus cereus mm7 and expressing them in E. coli. Journal of Investigative Genomics. 2017; 4(1): 7-14.

19. Lin JH, Lee MC, Sue YS, Liu YC, Li SY. Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly (3-hydroxybutyrate) (PHB) production. Applied Microbiology and Biotechnology. 2017; 101: 6419-6430.

20. Nayak DS, Singh B. Identification and isolation of acetoacetyl-CoA reducatase (phbB) gene involved in poly (β-hydroxybutyrate) biosynthesis in Ralstonia eutropha (IMTCC 1954 strain).‏ International Journal of Current Microbiology and Applied Sciences. 2018; 7: 349-355.

21. Wu H, Chen J, Chen GQ. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli. Applied Microbiology and Biotechnology. 2016; 100(23): 9907-9916.‏

22. Deepa S, Bhuvanehswari, P, Kanimozhi K, Panneerselvam A. Utilization of industrial waste for the production of poly- beta- hydroxy butyrate (Phb) by Alcaligenes eutrophus. Research Journal of Science and Technology. 2013; 5(4): 421-424.

23. Alagunachiyar M, Jeevitha R, Handique M, Kesavan S.. Isolation and screening of poly hydroxy butyrate (PHB) producing bacteria from Jeppiaar Salt Pane, Chennai. Research Journal of Pharmacy and Technology. 2015; 8(9): 1276-1280.‏

24. Ojha N, Das N. Optimization and characterization of polyhydroxyalkanoates and its copolymers synthesized by isolated yeasts. Research Journal of Pharmacy and Technology. 2017; 10(3): 861-868.

25. ‏Reddy AR, Kumar RB, Prabhakar KV. Isolation and identification of polyhydroxybutyrate (PHB) producing bacteria from sewage sample. Research Journal of Pharmacy and Technology. (2017); 10(4): 1065-1069.‏

26. Jaiganesh R, Tattwaprakash C, Kumar GR, Iyappan S, Jaganathan MK. Characterization and optimization of process parameter for maximum Poly (− β-) hydroxyl butyrate production by bacteria isolated from the soil. Research Journal of Pharmacy and Technology. 2019; 12(10): 4926-4930.‏

27. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press‏, Cold Spring Harbor, New York, USA. 1989: 2^nd ed.

28. Sambrook J and Russell DW. Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 2001: 3^rd ed.

29. Schägger H, Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry. 1987; 166(2): 368-379.‏

30. Wu W, Welsh MJ. Rapid Coomassie blue staining and destaining of polyacrylamide gels. BioTechniques. 1996; 20(3), 386-388.‏

31. Hussain N, Jim EY, Muhassinv PM, Sherin CS. Cloning of PA-1L gene from Pseudomonas aeruginosa in E. coli. Research Journal of Engineering and Technology. 2013; 4(4): 187-190.‏

32. Mishra N, Khan RH, Dwivedi B, Mishra SK. Cloning of Gene Coding Glyceraldehyde 3-Phosphate Dehydrogenase. Asian Journal of Pharmacy and Technology. 2014; 4(4): 184-188.‏

33. Sadhukhan K, Dholakia D, Bhadalkar AL. Genomic Studies of Symbiodinium spp. to understand Coral Reef Resilience in India-A review. Research Journal of Science and Technology. 2018; 10(2): 157-164.‏

34. Hadi HW. Genetic study on Bacillus cereus. Research Journal of Pharmacy and Technology. 2019; 12(3): 1301-1306.‏

35. Sutar DA, Jain BU, Kondawar M. A Review Article on Study of Cloning. Asian Journal of Research in Pharmaceutical Science. 2019; 9(2): 148-150.‏

36. Li R, Zhang H, Qi Q. The production of polyhydroxyalkanoates in recombinant Escherichia coli. Bioresource Technology. 2007; 98(12): 2313-2320.‏

37. Li H, Zhou S, Johnson T, Vercruysse K, Lizhi O, Ranganathan P, Phambu N, Ropelewski AJ, Thannhauser TW. Genome structure of Bacillus cereus tsu1 and genes involved in cellulose degradation and Poly-3-Hydroxybutyrate synthesis. International Journal of Polymer Science. 2017; 2017: 1-12.‏

38. Rehman RA. Cloning and Sequencing of the Polyhydroxybutyrate (PHB) Gene from locally isolated Bacillus spp. (Doctoral dissertation, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan). 2012.

39. Galehdari H, Alaei S, Mirzaei M. Cloning of poly (3-Hydroxybutyrate) synthesis genes from Azotobacter vinelandii into Escherichia coli.‏ Jundishapur Journal of Microbiology. 2009; 2: 31-35.

40. Li ZJ, Cai L, Wu Q, Chen GQ. Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly (3-hydroxybutyrate) production. Applied Microbiology and Biotechnology. 2009; 83(5): 939-947.‏

41. Chien CC, Hong CC, Soo PC, Wei YH, Chen SY, Cheng ML, Sun YM. Functional expression of phaCAB genes from Cupriavidus taiwanensis strain 184 in Escherichia coli for polyhydroxybutyrate production. Applied Biochemistry and Biotechnology. 2010; 162(8): 2355-2364.

42. Clemente T, Shah D, Tran M, Stark D, Padgette S, Dennis D, Brückener K, Steinbüchel A, Mitsky T. Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems. Applied Microbiology and Biotechnology. 2000; 53(4): 420-429.

43. Desetty RD, Mahajan VS, Khan BM, Rawal SK. Isolation and heterologous expression of PHA synthesising genes from Bacillus thuringiensis R1. World Journal of Microbiology and Biotechnology. 2008; 24(9): 1769-1774.

44. Tomizawa S, Hyakutake M, Saito Y, Agus J, Mizuno K, Abe H, Tsuge T. Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli. Biomacromolecules. 2011; 12(7): 2660-2666.‏

45. Tanaka T, Shima Y, Ogawa N, Nagayama K, Yoshida T, Ohmachi, T. Expression, identification and purification of Dictyostelium acetoacetyl-CoA thiolase expressed in Escherichia coli. International Journal of Biological Sciences. 2011; 7(1): 9.-17.

46. Shi H, Kyuwa K, Takasu M, Shimizu, K. Temperature-induced expression of phb genes in Escherichia coli and the effect of temperature patterns on the production of poly-3-hydroxybutyrate. Journal of Bioscience and Bioengineering. 2001; 91(1): 21-26.‏

Received on 10.09.2020 Modified on 02.10.2020

Research J. Pharm. and Tech. 2021; 14(6):3299-3306.

DOI: 10.52711/0974-360X.2021.00574